P386: CELL FATE DETERMINING TRANSCRIPTION FACTORS AS VULNERABILITIES IN CHILDHOOD AND ADULT MLL-REARRANGED AND T(8;16) ACUTE MYELOID LEUKEMIA
Authors: S. Arza Apalategi, B. Heuts, S. Bergevoet, S. van Heeringen, J. Jansen, J. Martens, B. van der Reijden
Journal: HemaSphere (2022)

Background: The MLL-AF9 protein contributes to acute myeloid leukemia (AML) development through transcriptional activation of oncogenic gene programs. Remarkably, MLL-AF9 AML consists of two subgroups, one with high oncogenic MECOM expression and dismal outcome; the other with a unique gene expression profile lacking MECOM expression and a favourable outcome. How the differences between these two clinically and biologically different subgroups are brought about in the context of the same MLL-AF9 genetic background is currently unknown. Aims: To identify vulnerabilities in AML beyond the MLL-AF9 oncofusion gene we determined cell fate determining transcription factors pertinent to the two clinically and biologically different subgroups. Methods: Transcription factors key to cell fate were determined by first analysing differential transcriptional start sites of genes and gene enhancers by genome-wide “Cap Analysis Gene Expression (CAGE)”-sequencing of three primary MECOM positive and negative MLL-AF9 AML samples. Sequencing data were subsequently analysed using a new bioinformatic tool that builds gene regulatory networks based on differential gene expression and enhancer activities to predict key transcription factors in cell fate determination. For predicted factors AML cohort-wide gene expression patterns, transforming potential and downstream gene programs were determined. Results: Bioinformatic analyses of CAGE-seq data identified the transcription factor MECOM as top hit for cell fate determination in MECOM+ MLL-AF9, confirming the power of this approach. In the group lacking MECOM expression we identified HMX3 as most influential transcription factor. HMX3 is essential for neuronal development and not expressed in any healthy blood cell type. In a large adult and childhood AML cohort (totalling 630 cases), high HMX3 expression was observed in 4% and 11% cases, respectively. HMX3 positive cases clustered tightly together in unsupervised clustering analyses, and 80% of cases were positive for MLL-rearranged (MLL-AF9, AF10, ENL) and t(8;16) AML. None were positive for other known recurrent AML mutations except for RAS mutations. In line with these observations, MLL-AF9 positive human cell lines expressed HMX3 (eg. THP1 and MOLM13). HMX3 silencing in THP1 and MOLM13 cells caused pronounced cell cycle arrest and apoptosis evidenced by PI, Annexin and 7AAD staining in flow cytometry. Forced lentiviral HMX3 expression in primary human GCSF-mobilized CD34+ cells resulted in an over 70% clonogenic growth inhibition compared to control transduced cells. To identify relevant gene programs we silenced HMX3 in MOLM13 cells followed by RNA-sequencing. Gene ontology analyses of over 3000 differentially expressed genes (p<0.01) suggested a role for HMX3 in cell cycle progression, sister chromatid separation, and immune response suppression (p<0.05). Summary/Conclusion: Bioinformatic analyses of CAGE-sequencing of primary leukemia samples predicted the transcription factors MECOM and HMX3 key to poor and good outcome MLL-AF9 AML, respectively. High HMX3 expression associated with a unique gene expression profile and associated strongly with MLL-rearranged and t(8;16) AML. HMX3 only was unable to transform healthy human immature blood cells but appeared essential for survival of MLL-AF9 leukemia cell lines, potentially by positively regulating cell cycle progression and immune evasion. Insight into the factors that activate HMX3 expression in AML could provide potential targets for therapies in MLL-rearranged and t(8;16) AML.